3D living neural networks

项目摘要/摘要 精神疾病，包括精神分裂症、抑郁症和自闭症谱系障碍，仍然很差 可以理解，尽管很明显，它们主要代表皮质障碍。大脑皮质是大脑的主要部位 更高的精神功能，然而，尽管进行了广泛的研究，但关于皮质如何工作仍然没有统一的理论。 这在一定程度上是因为神经科学家所需的工具有限，无法进行精确的、可重复的研究 神经回路的形成以及控制可塑性和修复的机制的研究。大多数研究 到目前为止完成的工作依赖于2D细胞培养或对活脑的研究。控制蜂窝分布的一些能力 直接连接神经细胞。然而，细胞随后被洗到底物上。而单元格 通常附着在有图案的表面上，没有控制细胞分布的机制 单元格分辨率。其他技术已经研究了带有“笼子”的平面电极阵列栅格的使用。 这只允许在每个笼子里存放一个细胞。这提高了明确映射的能力 检测特定神经元的信号，并将刺激限制在单个细胞上。然而，这种方法并不是 易于扩展到3D环境，而金属衬底不能准确地模拟自然细胞 环境，这可以改变细胞的行为。或者，使用光刺激和光刺激对脑片进行研究 钙成像可以绕过这些问题中的许多。然而，这种方法提供了令人望而生畏的 复杂性使得神经科学家要解开大脑的功能具有挑战性。 我们的方法为神经科学研究提供了几个关键的好处。通过利用最近的 钙成像和光刺激的进展我们消除了整个神经对电极的需要 网络，以刺激和探测连接。因此，纯水凝胶支架可以用作 支持结构，并作为引导神经生长的通道的来源。立体平版印刷使用户能够 为了快速定义聚合物网络的形状，分步重复方法能够实现任意结构 X和y尺寸，以及附加分层，可实现大规模轴向尺寸。此外，通过合并 已经实现了。我们的商用光学诱捕系统能够操纵数百个物体 同时，以高速和亚细胞分辨率。当组合光学捕获系统时 有了立体光刻术，完整的解决方案将允许科学家研究生物过程 前所未有的速度、分辨率和重复性。 博尔德非线性系统公司和科罗拉多大学提议将他们在建筑方面的专业知识结合起来 SLM和SLM显微镜两个阶段的项目，最终目标是制造动态3D组织 支架制造在神经科学和临床研究中是一个实际的现实。在第一阶段，我们计划建立一个 紧凑、廉价、用户友好的倒置显微镜，带有用于光学捕获的模块，以及 立体平版印刷。该设备将自动对准并与适当的软件集成，以便它可以 开箱即用，在几个神经生物学项目中应用，包括研究 可塑性和修复、药物和毒素筛选、化学和生物传感、生物相容性测试 假体与人体的接口和神经连接再生的研究 治疗脊髓损伤。在第二阶段，我们将增加制造系统的生产能力，并延长 系统的自动化。最终的目标是设计一种能够制造大规模神经的工具 具有微米分辨率的网络和组织支架，无需用户控制。此外，在第二阶段，BNS将 与奥林巴斯合作，为现有的光刻设备设计“插入式”光学陷印和立体光刻模块 奥林巴斯的显微镜。这将为拟议的研究提供一个既定的分销渠道，并将 允许用户利用专门针对其个人研究的现有成像方式。

Project Summary/Abstract Mental disease, including schizophrenia, depression and autism spectrum disorders, are still poorly understood, although it is clear that they mostly represent cortical disorders. The cortex is the primary site of higher mental functions, yet despite extensive research there is still no unified theory of how the cortex works. This is partly due to the fact that neuroscientists have limited tools required for precise repeatable studies of neural circuit formation and for studies of the mechanisms that control plasticity and repair. Most research completed to date relies on 2D cell cultures or studies of live brains. Some ability to control cellular distribution within 2D cultures has been demonstrated by patterning an extra-cellular protein, for example polylysine, to direct neuronal cell attachment. However, cells are subsequently washed onto the substrate. While cells generally adhere to the patterned surfaces, there is no mechanism for controlling cellular distribution with single cell resolution. Other techniques have investigated use of grids of planar electrode arrays with "cages" that only allow a single cell to be deposited in each cage. This improved the ability to unambiguously map detected signals to specific neurons, and to confine stimulation to single cells. However, this approach is not easily scalable to 3D environments, and metallic substrates do not accurately mimic a cells natural environment, which can alter cell behavior. Alternatively, studies of brain slices using photostimulation and calcium imaging circumvent many of these problems. However, this approach presents a daunting level of complexity making it challenging for neuroscientists to unravel function of the brain. Our approach offers several key benefits to neuroscience research. By taking advantage of recent advances in calcium imaging and photostimulation we remove the need for electrodes throughout our neural network to stimulate and probe connectivity. As a result a purely hydrogel scaffold can be used as the supporting structure, and as the source of channels to direct neural growth. Stereolithography enables the user to rapidly define the shape of the polymer network, step and repeat methods enable structures of arbitrary dimensions in x and y, and additive layering enables large scale axial dimensions. Furthermore, by merging stereolithography with optical trapping, micron scale control of the position of cells within the polymer structure is realized. Our commercially available optical trapping system is capable of manipulating hundreds of objects simultaneously, at high speed, and with sub-cellular resolution. When the optical trapping system is combined with stereolithography, the complete solution will allow scientists to study biological processes with unprecedented speed, resolution, and repeatability. Boulder Nonlinear Systems and the University of Colorado propose to combine their expertise in building SLMs and in SLM microscopy in a two-phase project with the ultimate goal of making dynamic 3D tissue scaffold fabrication a practical reality in neuroscience and clinical research. In the first phase we plan to build a compact, inexpensive, user-friendly inverted microscope with modules for optical trapping, and stereolithography. The device will be self-aligning and integrated with appropriate software so that it can be used, out of the box, for applications in several neurobiological projects including studies of mechanisms for plasticity and repair, drug and toxin screening, chemical and biological sensing, biocompatibility tests at the interface between a prosthetic device and human body, and research into regeneration of nerve connections for spinal cord injuries. In Phase II we will increase the throughput of the fabrication system, and extend the automation of the system. The ultimate goal is to design a tool capable of fabricating large scale neural networks and tissue scaffolds with micron resolution free of user control. Additionally in the Phase II BNS will collaborate with Olympus to design "bolt-on" optical trapping and stereolithography modules for existing Olympus microscopes. This will provide an established distribution channel for the proposed research and will allow users to utilize existing imaging modalities specialized to their individual studies.